Methods and compositions for treating brain diseases

ABSTRACT

The present disclosure provides methods of treating a disease or delivering a therapeutic agent to a mammal comprising administering to the mammal&#39;s cisterna magna and/or ventricle an rAAV particle containing a vector comprising a nucleic acid encoding a therapeutic protein inserted between a pair of AAV inverted terminal repeats in a manner such that cells with access to the cerebrospinal fluid (CSF) express the therapeutic agent and in certain embodiments secretes the therapeutic agent into the CSF for distribution to the brain.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/907,776, filed Jan. 26, 2016, which is a U.S.national stage application of International Patent Application No.PCT/US2014/047338, filed Jul. 20, 2014, which claims priority to U.S.Provisional Patent Application No. 61/859,157, filed Jul. 26, 2013. Theentirety of the applications is hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 13, 2014, isnamed 17023_139WO1_SL.txt and is 30,720 bytes in size.

BACKGROUND

Gene transfer is now widely recognized as a powerful tool for analysisof biological events and disease processes at both the cellular andmolecular level. More recently, the application of gene therapy for thetreatment of human diseases, either inherited (e.g., ADA deficiency) oracquired (e.g., cancer or infectious disease), has received considerableattention. With the advent of improved gene transfer techniques and theidentification of an ever expanding library of defective gene-relateddiseases, gene therapy has rapidly evolved from a treatment theory to apractical reality.

Traditionally, gene therapy has been defined as a procedure in which anexogenous gene is introduced into the cells of a patient in order tocorrect an inborn genetic error. Although more than 4500 human diseasesare currently classified as genetic, specific mutations in the humangenome have been identified for relatively few of these diseases. Untilrecently, these rare genetic diseases represented the exclusive targetsof gene therapy efforts. Accordingly, most of the NIH approved genetherapy protocols to date have been directed toward the introduction ofa functional copy of a defective gene into the somatic cells of anindividual having a known inborn genetic error. Only recently, haveresearchers and clinicians begun to appreciate that most human cancers,certain forms of cardiovascular disease, and many degenerative diseasesalso have important genetic components, and for the purposes ofdesigning novel gene therapies, should be considered “geneticdisorders.” Therefore, gene therapy has more recently been broadlydefined as the correction of a disease phenotype through theintroduction of new genetic information into the affected organism.

In in vivo gene therapy, a transferred gene is introduced into cells ofthe recipient organism in situ that is, within the recipient. In vivogene therapy has been examined in several animal models. Several recentpublications have reported the feasibility of direct gene transfer insitu into organs and tissues such as muscle, hematopoietic stem cells,the arterial wall, the nervous system, and lung. Direct injection of DNAinto skeletal muscle, heart muscle and injection of DNA-lipid complexesinto the vasculature also has been reported to yield a detectableexpression level of the inserted gene product(s) in vivo.

Treatment of diseases of the central nervous system, e.g., inheritedgenetic diseases of the brain, remains an intractable problem. Examplesof such are the lysosomal storage diseases and Alzheimer's disease.Collectively, the incidence of lysosomal storage diseases (LSD) is 1 in10,000 births world wide, and in 65% of cases, there is significantcentral nervous system (CNS) involvement. Proteins deficient in thesedisorders, when delivered intravenously, do not cross the blood-brainbarrier, or, when delivered directly to the brain, are not widelydistributed. Thus, therapies for the CNS deficits need to be developed.

SUMMARY

The present invention provides a method of delivering a therapeuticagent (e.g., protein or nucleic acid) to the central nervous system of amammal, comprising administering to the mammal's cisterna magna an rAAVparticle comprising an AAV capsid protein and a vector comprising anucleic acid encoding a therapeutic agent inserted between a pair of AAVinverted terminal repeats in a manner effective to infect cells thatcontact the cerebrospinal fluid (CSF) of in the mammal such that thecells express the therapeutic agent in the mammal.

The present invention provides a method of treating a disease in amammal comprising administering to the mammal's cisterna magna an rAAVparticle comprising an AAV capsid protein and a vector comprising anucleic acid encoding a therapeutic agent (e.g., a therapeutic nucleicacid or a nucleic acid encoding a protein) inserted between a pair ofAAV inverted terminal repeats in a manner effective to infect cells thatcontact the cerebrospinal fluid (CSF) in the mammal, wherein the cellexpresses the therapeutic agent so as to treat the disease.

In certain embodiments, the AAV particle is an rAAV2 particle. As usedherein, the term AAV2/1 is used to mean an AAV2 ITR and AAV1 capsid, theterm AAV2/2 is an AAV2 ITR and AAV2 capsid, the term AAV2/4 is an AAV2ITR and AAV4 capsid, etc. In certain embodiments, the AAV particle is anrAAV8 particle. In certain embodiments, the AAV particle is an rAAV9particle. In certain embodiments, the AAV particle is an rAAVrh10particle. In certain embodiments, the rAAV capsid has at least 80%homology to AAV2 capsid protein VP1, VP2, and/or VP3. In certainembodiments, the rAAV2 capsid has 100% homology to AAV2 capsid VP1, VP2,and/or VP3. In certain embodiments, the rAAV capsid has at least 80%homology to AAV4 capsid protein VP1, VP2, and/or VP3. In certainembodiments, the rAAV4 capsid has 100% homology to AAV4 capsid VP1, VP2,and/or VP3. In certain embodiments, the rAAV capsid has at least 80%homology to AAV9 capsid protein VP1, VP2, and/or VP3. In certainembodiments, the rAAV9 capsid has 100% homology to AAV9 capsid VP1, VP2,and/or VP3.

In certain embodiments, the rAAV particle is an rAAV2 particle thatinfects the non-rodent ependymal cell at an rate of more than 20% thanthe infectivity rate of AAV4, such as at a rate of more than 50% or100%, 1000% or 2000% than the infectivity rate of AAV4.

In certain embodiments, the cell expresses the therapeutic agent andsecretes the therapeutic agent into the CSF. In certain embodiments, thecell is an ependymal, pial, endothelial or meningeal cell. In certainembodiments, the method further comprises additionally administering therAAV to the non-human primate's brain ventricle, subarachnoid spaceand/or intrathecal space.

The present invention provides a method of delivering a nucleic acid toa brain cell of a mammal comprising administering to the brain cell anAAV particle containing a vector comprising the nucleic acid insertedbetween a pair of AAV inverted terminal repeats, thereby delivering thenucleic acid to the brain cell. In certain embodiments, the rAAV is anrAAV2 particle that infects the brain cell at an rate of more than 20%than the infectivity rate of AAV4, such as at a rate of more than 50% or100%, 1000% or 2000% than the infectivity rate of AAV4.

In certain embodiments, the disease is a lysosomal storage disease(LSD). In certain embodiments, the LSD is infantile or late infantileceroid lipofuscinoses, neuronopathic Gaucher, Juvenile Batten, Fabry,MLD, Sanfilippo A, Hunter, Krabbe, Morquio, Pompe, Niemann-Pick C,Tay-Sachs, Hurler (MPS-I H), Sanfilippo B, Maroteaux-Lamy, Niemann-PickA, Cystinosis, Hurler-Scheie (MPS-I H/S), Sly Syndrome (MPS VII), Scheie(MPS-I S), Infantile Batten, GM1 Gangliosidosis, Mucolipidosis typeII/III, or Sandhoff disease. In certain embodiments, the disease isLINCL. In certain embodiments, the disease is a neurodegenerativedisease, such as Alzheimer's disease, Huntington's disease, ALS,hereditary spastic hemiplegia, primary lateral sclerosis, spinalmuscular atrophy, Kennedy's disease, a polyglutamine repeat disease, orParkinson's disease.

In certain embodiments, the mammal is a non-rodent mammal, such as aprimate, horse, sheep, goat, pig, or dog. In certain embodiments, theprimate is a human.

In certain embodiments, the therapeutic agent is a therapeutic nucleicacid. In certain embodiments, the therapeutic agent is a protein.

In certain embodiments, the nucleic acid encodes a lysosomal hydrolase.In certain embodiments, the nucleic acid encodes TPP1.

In certain embodiments, the therapeutic protein is a protective ApoEisoform protein. As used herein, the term “protective ApoE isoform” isused to distinguish ApoE isoforms that decrease the risk of Alzheimer'sdisease by at least 5%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100% or more.

In certain embodiments, the protective ApoE isoform has at least about80% homology to ApoE ϵ2. In certain embodiments, the protective ApoEisoform has 100% homology to ApoE ϵ2.

In certain embodiments, the rAAV particle is injected at 1-3 locationsin the brain, such as at one, two, or three locations in the brain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C together are an alignment of AAV2 (SEQ ID NO:1) and AAV4(SEQ ID NO:2) proteins and FIGS. 1D-1J together are an alignment of AAV2(SEQ ID NO:3) and AAV4 (SEQ ID NO:4) nucleotides based on the sequencefrom AAV2 (NC_001401) and AAV4 (NC_001829).

FIG. 2 shows an illustration of “cross correction” between cells. Sandsand Davidson,

Mol Ther 13(5):839-849, 2006.

FIG. 3. Top: Immunohistochemical staining for human TPP1 after AAV2mediated delivery into a LINCL dog model that is deficient in canineTPP 1. Left, treated dog. Right, untreated deficient animal. Compare thestrong positive staining on the left to the background staining in theright panel. Bottom. Western blot for TPP1 showing the presence of humanTPP1 in the treated, deficient (LINCL) dog. Both normal and deficientdogs do not show the presence of the band, as they do not express humanTPP1.

FIG. 4A. Microphotographs showing the representative autofluorescencedepictive of the pathological accumulation of lipofuscin in the neuronalceroid lipofuscinoses. Left panel, autofluorescence in an AAV2.TPP1treated LINCL dog. Right panel, autofluoresence in a control, untreatedLINCL dog. Note the reduction in autofluorence with therapy.

FIG. 4B. MRI scans of an untreated LINCL dog (upper left), a untreatednormal dog (upper right), and two AAV2.TPP1 treated dogs (lower panels).The volumes of vector delivered are indicated in the lower left of thebottom panels. The viral titer was approximately 1e13 genomes/mL.

FIG. 4C. Volumetric reconstructions of the ventricles of the dogs imagedin 4B (left panels). The graph in the right panel denotes the volumesfrom the images in the left panels. Note the extensive reduction inventricular volume even with these low doses of vector (stated in FIG.4B legend).

FIG. 4D. Immunohistochemical staining in varying brain regions showsextensive distribution of TPP1 protein after AAV.TPP1 gene transfer tothe ventricular system of the LINCL dog. Top panels are coronal sectionsfrom the dog brain atlas; the lower right insets present the sagittalview of the coronal image. The immunohistochemically stained sectionsbelow the panels from the atlas show the extent of staining in sectionsfrom those regions. Together the data show extensive distribution ofenzyme.

FIG. 5. huTPP1 enzyme activity in CSF following AAV.TPP1 deliverydeclined shortly after viral gene transfer. Left panel: TPP1 activity inCSF in treated Animals Co and S exceeds normal activity levels very soonafter AAV.TPP1 gene transfer, and then rapidly declines to undetectablelevels. Animals N, Po and Pi are normal or heterozygous dogs and areshown for reference only of the range of TPP1 activity levels inclinically normal dogs.

FIG. 6 shows the results of pre-treating with mycophenolate on providingfor sustained activity.

FIG. 7. Introduction of mycophenolate at the time of enzyme activitydecline, or prior to gene transfer, dramatically improves the durabilityof TPP1 expression in dog after AAV.TPP1 delivery to ependyma. Left andright upper graphs: Enzyme activity as a function of time. Alsoindicated is the time at which mycophenolate was administered. Note thehigh and sustained levels after recover from loss of expression inAnimals SR and B, and the extremely high sustained levels in Animal F.Thus, mycophenolate pre-treatment in animals null for recombinantprotein helps provide for sustained gene expression in transduced braincells. Lower graph: Expansion from the upper right graph to demonstratethat there is enzyme over and above background levels, and close tonormal levels or above (0.1-0.4 pmol/mg).

FIG. 8. Sustained enzyme expression in CSF elevates interstitial levelsof enzyme. Enzyme activity in various brain regions is above normal.

FIGS. 9A and 9B. Immunohistochemical staining in varying brain regionsshows extensive distribution of TPP1 protein after AAV.TPP1 genetransfer to the ventricular system of the LINCL dog. Representativepanels from the dog brain atlas show the region of the brain beingevaluated, which is also depicted by the line. Together the data showextensive distribution of enzyme.

FIG. 10 AAV.TPP1 gene therapy delays the onset of disease phenotypes(appearance of the first red line on the left vs. the first blue line onthe left) and the progression of disease (the spacing of the red linesvs. the spacing of the blue lines). Animal life span was nearly doubledin some dogs, others are still under evaluation.

FIG. 11 Animals with sustained TPP1 secretion from ependymal showevidence of enzyme activity in peripheral organs and brain dura. For twoanimals, BG and SR, there was notable enzyme activity in brain dura andalso the liver.

FIG. 12 The approach used to provide clinical benefit to the LINCL dogis translatable to primates. Rhesus macaques were given anintraventricular injection of AAV2.TPP1 (1.5 mL of 1e13 vectorgenomes/mL) and TPP1 activity in brain stem (Medulla; left graph) andCSF (right graph) measured 3 months after gene transfer. These arenormal monkeys with normal levels of TPP1 activity (rangenoted—Control). In all but one animal, the enzyme activity exceeds thatof normal monkeys. Evidence of TPP1 activity in monkey brain 3 monthsafter gene transfer using immunohistochemistry staining against therecombinant human TPP1 expressed from the AAV vector.

FIG. 13 shows the vestibular area (brainstem) in the non-human primates.

FIG. 14 provides the Human TPP1 amino acid sequence (SEQ ID NO: 5).

FIGS. 15A and 15B together provide the Human TPP1 nucleic acid sequence(SEQ ID NO: 6).

FIG. 16 provides the Macaca mulatta TPP1 amino acid sequence (SEQ ID NO:7).

FIG. 17 provides the Macaca fascicularis TPP1 amino acid sequence (SEQID NO: 8).

DETAILED DESCRIPTION

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family. AAV is distinct from the other members of thisfamily by its dependence upon a helper virus for replication. In theabsence of a helper virus, AAV may integrate in a locus specific mannerinto the q arm of chromosome 19. The approximately 5 kb genome of AAVconsists of one segment of single stranded DNA of either plus or minuspolarity. The ends of the genome are short inverted terminal repeatswhich can fold into hairpin structures and serve as the origin of viralDNA replication. Physically, the parvovirus virion is non-enveloped andits icosohedral capsid is approximately 20 nm in diameter.

To-date numerous serologically distinct AAVs have been identified, andmore than a dozen have been isolated from humans or primates. The genomeof AAV2 is 4680 nucleotides in length and contains two open readingframes (ORFs). The left ORF encodes the non-structural Rep proteins, Rep40, Rep 52, Rep 68 and Rep 78, which are involved in regulation ofreplication and transcription in addition to the production ofsingle-stranded progeny genomes. Furthermore, two of the Rep proteinshave been associated with the preferential integration of AAV genomesinto a region of the q arm of human chromosome 19. Rep68/78 has alsobeen shown to possess NTP binding activity as well as DNA and RNAhelicase activities. The Rep proteins possess a nuclear localizationsignal as well as several potential phosphorylation sites. Mutation ofone of these kinase sites resulted in a loss of replication activity.

The ends of the genome are short inverted terminal repeats (ITR) whichhave the potential to fold into T-shaped hairpin structures that serveas the origin of viral DNA replication. Within the ITR region twoelements have been described which are central to the function of theITR, a GAGC repeat motif and the terminal resolution site (trs). Therepeat motif has been shown to bind Rep when the ITR is in either alinear or hairpin conformation. This binding serves to position Rep68/78for cleavage at the trs which occurs in a site- and strand-specificmanner. In addition to their role in replication, these two elementsappear to be central to viral integration. Contained within thechromosome 19 integration locus is a Rep binding site with an adjacenttrs. These elements have been shown to be functional and necessary forlocus specific integration.

The AAV virion is a non-enveloped, icosohedral particle approximately 25nm in diameter, consisting of three related proteins referred to as VP1,VP2 and VP3. The right ORF encodes the capsid proteins VP1, VP2, andVP3. These proteins are found in a ratio of 1:1:10 respectively and areall derived from the right-hand ORF. The capsid proteins differ fromeach other by the use of alternative splicing and an unusual startcodon. Deletion analysis has shown that removal or alteration of VP1which is translated from an alternatively spliced message results in areduced yield of infections particles. Mutations within the VP3 codingregion result in the failure to produce any single-stranded progeny DNAor infectious particles. An AAV particle is a viral particle comprisingan AAV capsid protein. An AAV capsid polypeptide can encode the entireVP1, VP2 and VP3 polypeptide. The particle can be a particle comprisingAAV2 and other AAV capsid proteins (i.e., a chimeric protein, such asAAV4 and AAV2). Variations in the amino acid sequence of the AAV2 capsidprotein are contemplated herein, as long as the resulting viral particlecomprises the AAV2 capsid remains antigenically or immunologicallydistinct from AAV4, as can be routinely determined by standard methods.Specifically, for example, ELISA and Western blots can be used todetermine whether a viral particle is antigenically or immunologicallydistinct from AAV4. Furthermore, the AAV2 viral particle preferablyretains tissue tropism distinct from AAV4.

An AAV2 particle is a viral particle comprising an AAV2 capsid protein.An AAV2 capsid polypeptide encoding the entire VP1, VP2, and VP3polypeptide can overall have at least about 63% homology (or identity)to the polypeptide having the amino acid sequence encoded by nucleotidesset forth in SEQ ID NO:1 (AAV2 capsid protein). The capsid protein canhave about 70% homology, about 75% homology, 80% homology, 85% homology,90% homology, 95% homology, 98% homology, 99% homology, or even 100%homology to the protein set forth in SEQ ID NO:1. The capsid protein canhave about 70% identity, about 75% identity, 80% identity, 85% identity,90% identity, 95% identity, 98% identity, 99% identity, or even 100%identity to the protein set forth in SEQ ID NO:1. The particle can be aparticle comprising another AAV and AAV2 capsid protein, i.e., achimeric protein. Variations in the amino acid sequence of the AAV2capsid protein are contemplated herein, as long as the resulting viralparticle comprising the AAV2 capsid remains antigenically orimmunologically distinct from AAV4, as can be routinely determined bystandard methods. Specifically, for example, ELISA and Western blots canbe used to determine whether a viral particle is antigenically orimmunologically distinct from AAV4. Furthermore, the AAV2 viral particlepreferably retains tissue tropism distinction from AAV4, such as thatexemplified in the examples herein, though an AAV2 chimeric particlecomprising at least one AAV2 coat protein may have a different tissuetropism from that of an AAV2 particle consisting only of AAV2 coatproteins.

As indicated in FIGS. 1A and 1B, AAV2 capsid sequence and AAV4 capsidsequence are about 60% homologous. In certain embodiments, the AAV2capsid comprises (or consists of) a sequence that is at least 65%homologous to the amino acid sequence set forth in SEQ ID NO:1.

In certain embodiments, the invention further provides an AAV2 particlecontaining, i.e., encapsidating, a vector comprising a pair of AAV2inverted terminal repeats. The nucleotide sequence of AAV2 ITRs is knownin the art. Furthermore, the particle can be a particle comprising bothAAV4 and AAV2 capsid protein, i.e., a chimeric protein. Moreover, theparticle can be a particle encapsidating a vector comprising a pair ofAAV inverted terminal repeats from other AAVs (e.g., AAV1-AAV9 andAAVrh10). The vector encapsidated in the particle can further comprisean exogenous nucleic acid inserted between the inverted terminalrepeats.

The following features of AAV have made it an attractive vector for genetransfer. AAV vectors have been shown in vitro to stably integrate intothe cellular genome; possess a broad host range; transduce both dividingand non dividing cells in vitro and in vivo and maintain high levels ofexpression of the transduced genes. Viral particles are heat stable,resistant to solvents, detergents, changes in pH, temperature, and canbe concentrated on CsC1 gradients or by other means. The presentinvention provides methods of administering AAV particles, recombinantAAV vectors, and recombinant AAV virions. For example, an AAV2 particleis a viral particle comprising an AAV2 capsid protein, or an AAV4particle is a viral particle comprising an AAV4 capsid protein. Arecombinant AAV2 vector is a nucleic acid construct that comprises atleast one unique nucleic acid of AAV2. A recombinant AAV2 virion is aparticle containing a recombinant AAV2 vector. To be considered withinthe term “AAV2 ITRs” the nucleotide sequence must retain one or bothfeatures described herein that distinguish the AAV2 ITR from the AAV4ITR: (1) three (rather than four as in AAV4) “GAGC” repeats and (2) inthe AAV2 ITR Rep binding site the fourth nucleotide in the first two“GAGC” repeats is a C rather than a T.

The promoter to drive expression of the protein or the sequence encodinganother agent to be delivered can be any desired promoter, selected byknown considerations, such as the level of expression of a nucleic acidfunctionally linked to the promoter and the cell type in which thevector is to be used. Promoters can be an exogenous or an endogenouspromoter. Promoters can include, for example, known strong promoterssuch as SV40 or the inducible metallothionein promoter, or an AAVpromoter, such as an AAV p5 promoter. Additional examples of promotersinclude promoters derived from actin genes, immunoglobulin genes,cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenoviralpromoters, such as the adenoviral major late promoter, an inducible heatshock promoter, respiratory syncytial virus, Rous sarcomas virus (RSV),etc.

The AAV vector can further comprise an exogenous (heterologous) nucleicacid functionally linked to the promoter. By “heterologous nucleic acid”is meant that any heterologous or exogenous nucleic acid can be insertedinto the vector for transfer into a cell, tissue or organism. Thenucleic acid can encode a polypeptide or protein or an antisense RNA,for example. By “functionally linked” is meant such that the promotercan promote expression of the heterologous nucleic acid, as is known inthe art, such as appropriate orientation of the promoter relative to theheterologous nucleic acid. Furthermore, the heterologous nucleic acidpreferably has all appropriate sequences for expression of the nucleicacid, as known in the art, to functionally encode, i.e., allow thenucleic acid to be expressed. The nucleic acid can include, for example,expression control sequences, such as an enhancer, and necessaryinformation processing sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.The nucleic acid can encode more than one gene product, limited only bythe size of nucleic acid that can be packaged.

The heterologous nucleic acid can encode beneficial proteins thatreplace missing or defective proteins required by the subject into whichthe vector in transferred or can encode a cytotoxic polypeptide that canbe directed, e.g., to cancer cells or other cells whose death would bebeneficial to the subject. The heterologous nucleic acid can also encodeantisense RNAs that can bind to, and thereby inactivate, mRNAs made bythe subject that encode harmful proteins. In one embodiment, antisensepolynucleotides can be produced from a heterologous expression cassettein an AAV viral construct where the expression cassette contains asequence that promotes cell-type specific expression.

Examples of heterologous nucleic acids which can be administered to acell or subject as part of the present AAV vector can include, but arenot limited to the nucleic acids encoding therapeutic agents, such aslysosomal hydrolases; tumor necrosis factors (TNF), such as TNF-alpha;interferons, such as interferon-alpha, interferon-beta, andinterferon-gamma; interleukins, such as IL-1, IL-1beta, and ILs-2through −14; GM-CSF; adenosine deaminase; secreted factors such asgrowth factors; ion channels; chemotherapeutics; lysosomal proteins;anti-apoptotic gene products; proteins promoting neural survival such asglutamate receptors and growth factors; cellular growth factors, such aslymphokines; soluble CD4; Factor VIII; Factor IX; T-cell receptors; LDLreceptor; ApoE; ApoC; alpha-1 antitrypsin; ornithine transcarbamylase(OTC); cystic fibrosis transmembrane receptor (CFTR); insulin; Fcreceptors for antigen binding domains of antibodies, such asimmunoglobulins; and antisense sequences which inhibit viralreplication, such as antisense sequences which inhibit replication ofhepatitis B or hepatitis non-A, non-B virus. Furthermore, the nucleicacid can encode more than one gene product, limited only by the size ofnucleic acid that can be packaged.

An AAV2 particle is a viral particle comprising an AAV2 capsid protein.Variations in the amino acid sequence of the AAV2 capsid protein arecontemplated herein, as long as the resulting viral particle comprisingthe AAV2 capsid remains antigenically or immunologically distinct fromAAV4, as can be routinely determined by standard methods. Specifically,for example, ELISA and Western blots can be used to determine whether aviral particle is antigenically or immunologically distinct from otherAAV serotypes.

The term “polypeptide” as used herein refers to a polymer of amino acidsand includes full-length proteins and fragments thereof. Thus, “protein”and “polypeptide” are often used interchangeably herein. Substitutionscan be selected by known parameters to be neutral. As will beappreciated by those skilled in the art, the invention also includesthose polypeptides having slight variations in amino acid sequences orother properties. Such variations may arise naturally as allelicvariations (e.g. due to genetic polymorphism) or may be produced byhuman intervention (e.g., by mutagenesis of cloned DNA sequences), suchas induced point, deletion, insertion and substitution mutants. Minorchanges in amino acid sequence are generally preferred, such asconservative amino acid replacements, small internal deletions orinsertions, and additions or deletions at the ends of the molecules.These modifications can result in changes in the amino acid sequence,provide silent mutations, modify a restriction site, or provide otherspecific mutations.

The present method provides a method of delivering a nucleic acid to acell comprising administering to the cell an AAV particle containing avector comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, thereby delivering the nucleic acid to thecell. Administration to the cell can be accomplished by any means,including simply contacting the particle, optionally contained in adesired liquid such as tissue culture medium, or a buffered salinesolution, with the cells. The particle can be allowed to remain incontact with the cells for any desired length of time, and typically theparticle is administered and allowed to remain indefinitely. For such invitro methods, the virus can be administered to the cell by standardviral transduction methods, as known in the art and as exemplifiedherein. Titers of virus to administer can vary, particularly dependingupon the cell type, but will be typical of that used for AAVtransduction in general. Additionally the titers used to transduce theparticular cells in the present examples can be utilized. The cells caninclude any desired cell in humans as well as other large (non-rodent)mammals, such as primates, horse, sheep, goat, pig, and dog.

More specifically, the present invention provides a method of deliveringa nucleic acid to a cell with contact to the circulating CSF, such as anependymal cell, a pial cell, meningeal cell, a brain endothelial cell,comprising administering to the cell an AAV particle containing a vectorcomprising the nucleic acid inserted between a pair of AAV invertedterminal repeats, thereby delivering the nucleic acid to the cell.

The present invention further provides a method of delivering a nucleicacid to a cell in a subject comprising administering to the subject anAAV particle comprising the nucleic acid inserted between a pair of AAVinverted terminal repeats, thereby delivering the nucleic acid to a cellin the subject.

Also provided is a method of delivering a nucleic acid to an ependymal,pial or other meningeal cell in a subject comprising administering tothe subject an AAV particle comprising the nucleic acid inserted betweena pair of AAV inverted terminal repeats, thereby delivering the nucleicacid to the ependymal, pial or other meningeal cell in the subject.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium targets brain vascular endothelium in a subjectthat has a disease, e.g., a lysosomal storage disease.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium targets brain vascular endothelium in a subjectthat does not have a lysosomal storage disease.

In certain embodiments, the viral vector comprises a nucleic acidsequence encoding a therapeutic agent. In certain embodiments, thetherapeutic agent is TPP1.

Certain embodiments of the present disclosure provide a cell comprisinga viral vector as described herein.

Certain embodiments of the present disclosure provide a method oftreating a disease in a mammal comprising administering a viral vectoror the cell as described herein to the mammal.

In certain embodiments, the mammal is human.

In certain embodiments, the disease is a lysosomal storage disease(LSD). In certain embodiments, the LSD is infantile or late infantileceroid lipofuscinoses, Gaucher, Juvenile Batten, Fabry, MLD, SanfilippoA, Late Infantile Batten, Hunter, Krabbe, Morquio, Pompe, Niemann-PickC, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B, Maroteaux-Lamy,Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I H/S), Sly Syndrome (MPSVII), Scheie (MPS-I S), Infantile Batten, GM1 Gangliosidosis,Mucolipidosis type IUIII, or Sandhoff disease.

In certain embodiments, the disease is a neurodegenerative disease. Incertain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Huntington's disease, ALS, hereditary spastic hemiplegia,primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, apolyglutamine repeat disease, or Parkinson's disease.

Certain embodiments of the present disclosure provide a method todeliver an agent to the central nervous system of a subject, comprisingadministering to the CSF with a viral vector described herein so thatthe transduced ependymal, pial, endothelial and/or other meningeal cellsexpress the therapeutic agent and deliver the agent to the centralnervous system of the subject. In certain embodiments, the viral vectortransduces ependymal, pial, endothelial and/or other meningeal cells.

Certain embodiments of the present disclosure provide a viral vector orcell as described herein for use in medical treatments.

Certain embodiments of the present disclosure provide a use of a viralvector or cell as described herein to prepare a medicament useful fortreating a disease, e.g., a lysosomal storage disease, in a mammal.

The vector may further comprise a lysosomal enzyme (e.g., a lysosomalhydrolase), a secreted protein, a nuclear protein, or a cytoplasmicprotein. As used herein, the term “secreted protein” includes anysecreted protein, whether naturally secreted or modified to contain asignal sequence so that it can be secreted.

Certain embodiments of the present disclosure provide a use of a viralvector or cell as described herein to prepare a medicament useful fortreating a disease, e.g., Alzheimer's disease, in a mammal.

The vector may further comprise a protective ApoE isoform protein. Asused herein, the term “secreted protein” includes any secreted protein,whether naturally secreted or modified to contain a signal sequence sothat it can be secreted. Nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. Additionally, multiple copies of the nucleic acidencoding enzymes may be linked together in the expression vector. Suchmultiple nucleic acids may be separated by linkers.

The present disclosure also provides a mammalian cell containing avector described herein. The cell may be human, and may be from brain.The cell type may be a stem or progenitor cell population.

The present disclosure provides a method of treating a disease such as agenetic disease or cancer in a mammal by administering a polynucleotide,polypeptide, expression vector, or cell described herein. The geneticdisease or cancer may be a lysosomal storage disease (LSD) such asinfantile or late infantile ceroid lipofuscinoses, Gaucher, JuvenileBatten, Fabry, MLD, Sanfilippo A, Late Infantile Batten, Hunter, Krabbe,Morquio, Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), SanfilippoB, Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-IH/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten, GM1Gangliosidosis, Mucolipidosis type II/III, or Sandhoff disease.

The genetic disease may be a neurodegenerative disease, such asHuntington's disease, ALS, hereditary spastic hemiplegia, primarylateral sclerosis, spinal muscular atrophy, Kennedy's disease,Alzheimer's disease, a polyglutamine repeat disease, or focal exposuresuch as Parkinson's disease.

Certain aspects of the disclosure relate to polynucleotides,polypeptides, vectors, and genetically engineered cells (modified invivo), and the use of them. In particular, the disclosure relates to amethod for gene or protein therapy that is capable of both systemicdelivery of a therapeutically effective dose of the therapeutic agent.

According to one aspect, a cell expression system for expressing atherapeutic agent in a mammalian recipient is provided. The expressionsystem (also referred to herein as a “genetically modified cell”)comprises a cell and an expression vector for expressing the therapeuticagent. Expression vectors include, but are not limited to, viruses,plasmids, and other vehicles for delivering heterologous geneticmaterial to cells. Accordingly, the term “expression vector” as usedherein refers to a vehicle for delivering heterologous genetic materialto a cell. In particular, the expression vector is a recombinantadenoviral, adeno-associated virus, or lentivirus or retrovirus vector.

The expression vector further includes a promoter for controllingtranscription of the heterologous gene. The promoter may be an induciblepromoter (described below). The expression system is suitable foradministration to the mammalian recipient. The expression system maycomprise a plurality of non-immortalized genetically modified cells,each cell containing at least one recombinant gene encoding at least onetherapeutic agent.

The cell expression system is formed in vivo. According to yet anotheraspect, a method for treating a mammalian recipient in vivo is provided.The method includes introducing an expression vector for expressing aheterologous gene product into a cell of the patient in situ, such asvia intravenous administration. To form the expression system in vivo,an expression vector for expressing the therapeutic agent is introducedin vivo into the mammalian recipient i.v., where the vector migrates viathe vasculature to the brain.

According to yet another aspect, a method for treating a mammalianrecipient in vivo is provided. The method includes introducing thetarget protein into the patient in vivo.

The expression vector for expressing the heterologous gene may includean inducible promoter for controlling transcription of the heterologousgene product. Accordingly, delivery of the therapeutic agent in situ iscontrolled by exposing the cell in situ to conditions, which inducetranscription of the heterologous gene.

The mammalian recipient may have a condition that is amenable to genereplacement therapy. As used herein, “gene replacement therapy” refersto administration to the recipient of exogenous genetic materialencoding a therapeutic agent and subsequent expression of theadministered genetic material in situ. Thus, the phrase “conditionamenable to gene replacement therapy” embraces conditions such asgenetic diseases (i.e., a disease condition that is attributable to oneor more gene defects), acquired pathologies (i.e., a pathologicalcondition which is not attributable to an inborn defect), cancers andprophylactic processes (i.e., prevention of a disease or of an undesiredmedical condition). Accordingly, as used herein, the term “therapeuticagent” refers to any agent or material, which has a beneficial effect onthe mammalian recipient. Thus, “therapeutic agent” embraces boththerapeutic and prophylactic molecules having nucleic acid or proteincomponents.

According to one embodiment, the mammalian recipient has a geneticdisease and the exogenous genetic material comprises a heterologous geneencoding a therapeutic agent for treating the disease. In yet anotherembodiment, the mammalian recipient has an acquired pathology and theexogenous genetic material comprises a heterologous gene encoding atherapeutic agent for treating the pathology. According to anotherembodiment, the patient has a cancer and the exogenous genetic materialcomprises a heterologous gene encoding an anti-neoplastic agent. In yetanother embodiment the patient has an undesired medical condition andthe exogenous genetic material comprises a heterologous gene encoding atherapeutic agent for treating the condition.

As used herein, the terms “a protective ApoE isoform,” “lysosomalenzyme,” a “secreted protein,” a “nuclear protein,” or a “cytoplasmicprotein” include variants or biologically active or inactive fragmentsof these polypeptides. A “variant” of one of the polypeptides is apolypeptide that is not completely identical to a native protein. Suchvariant protein can be obtained by altering the amino acid sequence byinsertion, deletion or substitution of one or more amino acid. The aminoacid sequence of the protein is modified, for example by substitution,to create a polypeptide having substantially the same or improvedqualities as compared to the native polypeptide. The substitution may bea conserved substitution. A “conserved substitution” is a substitutionof an amino acid with another amino acid having a similar side chain. Aconserved substitution would be a substitution with an amino acid thatmakes the smallest change possible in the charge of the amino acid orsize of the side chain of the amino acid (alternatively, in the size,charge or kind of chemical group within the side chain) such that theoverall peptide retains its spacial conformation but has alteredbiological activity. For example, common conserved changes might be Aspto Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu andSer to Cys, Thr or Gly. Alanine is commonly used to substitute for otheramino acids. The 20 essential amino acids can be grouped as follows:alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophanand methionine having nonpolar side chains; glycine, serine, threonine,cystine, tyrosine, asparagine and glutamine having uncharged polar sidechains; aspartate and glutamate having acidic side chains; and lysine,arginine, and histidine having basic side chains.

The amino acid changes are achieved by changing the codons of thecorresponding nucleic acid sequence. It is known that such polypeptidescan be obtained based on substituting certain amino acids for otheramino acids in the polypeptide structure in order to modify or improvebiological activity. For example, through substitution of alternativeamino acids, small conformational changes may be conferred upon apolypeptide that results in increased activity. Alternatively, aminoacid substitutions in certain polypeptides may be used to provideresidues, which may then be linked to other molecules to providepeptide-molecule conjugates which, retain sufficient properties of thestarting polypeptide to be useful for other purposes.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, wherein it is foundthat certain amino acids may be substituted for other amino acids havingsimilar hydropathic indices and still retain a similar biologicalactivity. Alternatively, substitution of like amino acids may be made onthe basis of hydrophilicity, particularly where the biological functiondesired in the polypeptide to be generated in intended for use inimmunological embodiments. The greatest local average hydrophilicity ofa “protein”, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity. Accordingly, it is noted thatsubstitutions can be made based on the hydrophilicity assigned to eachamino acid.

In using either the hydrophilicity index or hydropathic index, whichassigns values to each amino acid, it is preferred to conductsubstitutions of amino acids where these values are ±2, with ±1 beingparticularly preferred, and those with in ±0.5 being the most preferredsubstitutions.

The variant protein has at least 50%, at least about 80%, or even atleast about 90% but less than 100%, contiguous amino acid sequencehomology or identity to the amino acid sequence of a correspondingnative protein.

The amino acid sequence of the variant polypeptide correspondsessentially to the native polypeptide's amino acid sequence. As usedherein “correspond essentially to” refers to a polypeptide sequence thatwill elicit a biological response substantially the same as the responsegenerated by the native protein. Such a response may be at least 60% ofthe level generated by the native protein, and may even be at least 80%of the level generated by native protein.

A variant may include amino acid residues not present in thecorresponding native protein or deletions relative to the correspondingnative protein. A variant may also be a truncated “fragment” as comparedto the corresponding native protein, i.e., only a portion of afull-length protein. Protein variants also include peptides having atleast one D-amino acid.

The variant protein may be expressed from an isolated DNA sequenceencoding the variant protein. “Recombinant” is defined as a peptide ornucleic acid produced by the processes of genetic engineering. It shouldbe noted that it is well-known in the art that, due to the redundancy inthe genetic code, individual nucleotides can be readily exchanged in acodon, and still result in an identical amino acid sequence.

The present disclosure provides methods of treating a disease in amammal by administering an expression vector to a cell or patient. Forthe gene therapy methods, a person having ordinary skill in the art ofmolecular biology and gene therapy would be able to determine, withoutundue experimentation, the appropriate dosages and routes ofadministration of the expression vector used in the novel methods of thepresent disclosure.

According to one embodiment, the cells are transformed or otherwisegenetically modified in vivo. The cells from the mammalian recipient aretransformed (i.e., transduced or transfected) in vivo with a vectorcontaining exogenous genetic material for expressing a heterologous(e.g., recombinant) gene encoding a therapeutic agent and thetherapeutic agent is delivered in situ.

As used herein, “exogenous genetic material” refers to a nucleic acid oran oligonucleotide, either natural or synthetic, that is not naturallyfound in the cells; or if it is naturally found in the cells, it is nottranscribed or expressed at biologically significant levels by thecells. Thus, “exogenous genetic material” includes, for example, anon-naturally occurring nucleic acid that can be transcribed intoanti-sense RNA, as well as a “heterologous gene” (i.e., a gene encodinga protein which is not expressed or is expressed at biologicallyinsignificant levels in a naturally-occurring cell of the same type).

In the certain embodiments, the mammalian recipient has a condition thatis amenable to gene replacement therapy. As used herein, “genereplacement therapy” refers to administration to the recipient ofexogenous genetic material encoding a therapeutic agent and subsequentexpression of the administered genetic material in situ. Thus, thephrase “condition amenable to gene replacement therapy” embracesconditions such as genetic diseases (i.e., a disease condition that isattributable to one or more gene defects), acquired pathologies (i.e., apathological condition which is not attributable to an inborn defect),cancers and prophylactic processes (i.e., prevention of a disease or ofan undesired medical condition). Accordingly, as used herein, the term“therapeutic agent” refers to any agent or material, which has abeneficial effect on the mammalian recipient. Thus, “therapeutic agent”embraces both therapeutic and prophylactic molecules having nucleic acid(e.g., antisense RNA) and/or protein components.

Alternatively, the condition amenable to gene replacement therapy is aprophylactic process, i.e., a process for preventing disease or anundesired medical condition. Thus, the instant disclosure embraces acell expression system for delivering a therapeutic agent that has aprophylactic function (i.e., a prophylactic agent) to the mammalianrecipient.

In summary, the term “therapeutic agent” includes, but is not limitedto, agents associated with the conditions listed above, as well as theirfunctional equivalents. As used herein, the term “functional equivalent”refers to a molecule (e.g., a peptide or protein) that has the same oran improved beneficial effect on the mammalian recipient as thetherapeutic agent of which is it deemed a functional equivalent.

The above-disclosed therapeutic agents and conditions amenable to genereplacement therapy are merely illustrative and are not intended tolimit the scope of the instant disclosure. The selection of a suitabletherapeutic agent for treating a known condition is deemed to be withinthe scope of one of ordinary skill of the art without undueexperimentation.

AAV Vectors

In one embodiment, a viral vector of the disclosure is an AAV vector. An“AAV” vector refers to an adeno-associated virus, and may be used torefer to the naturally occurring wild-type virus itself or derivativesthereof. The term covers all subtypes, serotypes and pseudotypes, andboth naturally occurring and recombinant forms, except where requiredotherwise. As used herein, the term “serotype” refers to an AAV which isidentified by and distinguished from other AAVs based on capsid proteinreactivity with defined antisera, e.g., there are eight known serotypesof primate AAVs, AAV-1 to AAV-9 and AAVrh10. For example, serotype AAV2is used to refer to an AAV which contains capsid proteins encoded fromthe cap gene of AAV2 and a genome containing 5′ and 3′ ITR sequencesfrom the same AAV2 serotype. As used herein, for example, rAAV1 may beused to refer an AAV having both capsid proteins and 5′-3′ ITRs from thesame serotype or it may refer to an AAV having capsid proteins from oneserotype and 5′-3′ ITRs from a different AAV serotype, e.g., capsid fromAAV serotype 2 and ITRs from AAV serotype 5. For each exampleillustrated herein the description of the vector design and productiondescribes the serotype of the capsid and 5′-3′ ITR sequences. Theabbreviation “rAAV” refers to recombinant adeno-associated virus, alsoreferred to as a recombinant AAV vector (or “rAAV vector”).

An “AAV virus” or “AAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein (preferably by all of thecapsid proteins of a wild-type AAV) and an encapsidated polynucleotide.If the particle comprises heterologous polynucleotide (i.e., apolynucleotide other than a wild-type AAV genome such as a transgene tobe delivered to a mammalian cell), it is typically referred to as“rAAV”.

In one embodiment, the AAV expression vectors are constructed usingknown techniques to at least provide as operatively linked components inthe direction of transcription, control elements including atranscriptional initiation region, the DNA of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian cell. The resulting construct whichcontains the operatively linked components is flanked (5′ and 3′) withfunctional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. As used herein,an “AAV ITR” need not have the wild-type nucleotide sequence depicted,but may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV1, AAV2, AAV3,AAV4, AAV5, AAV7, etc. Furthermore, 5′ and 3′ ITRs which flank aselected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

In one embodiment, AAV ITRs can be derived from any of several AAVserotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5,AAV7, etc. Furthermore, 5′ and 3′ ITRs which flank a selected nucleotidesequence in an AAV expression vector need not necessarily be identicalor derived from the same AAV serotype or isolate, so long as theyfunction as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

In one embodiment, AAV capsids can be derived from AAV2. Suitable DNAmolecules for use in AAV vectors will be less than about 5 kilobases(kb), less than about 4.5 kb, less than about 4kb, less than about 3.5kb, less than about 3 kb, less than about 2.5 kb in size and are knownin the art.

In one embodiment, the selected nucleotide sequence is operably linkedto control elements that direct the transcription or expression thereofin the subject in vivo. Such control elements can comprise controlsequences normally associated with the selected gene. Alternatively,heterologous control sequences can be employed. Useful heterologouscontrol sequences generally include those derived from sequencesencoding mammalian or viral genes. Examples include, but are not limitedto, the SV40 early promoter, mouse mammary tumor virus LTR promoter;adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, polII promoters, pol III promoters, synthetic promoters, hybrid promoters,and the like. In addition, sequences derived from nonviral genes, suchas the murine metallothionein gene, will also find use herein. Suchpromoter sequences are commercially available from, e.g., Stratagene(San Diego, Calif.).

In one embodiment, both heterologous promoters and other controlelements, such as CNS-specific and inducible promoters, enhancers andthe like, will be of particular use. Examples of heterologous promotersinclude the CMV promoter. Examples of CNS-specific promoters includethose isolated from the genes from myelin basic protein (MBP), glialfibrillary acid protein (GFAP), and neuron specific enolase (NSE).Examples of inducible promoters include DNA responsive elements forecdysone, tetracycline, hypoxia and aufin.

In one embodiment, the AAV expression vector which harbors the DNAmolecule of interest bounded by AAV ITRs, can be constructed by directlyinserting the selected sequence(s) into an AAV genome which has had themajor AAV open reading frames (“ORFs”) excised therefrom. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.Such constructs can be designed using techniques well known in the art.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques. For example, ligations can be accomplished in 20 mM Tris-ClpH 7.5, 10 mM MgC₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, andeither 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for“sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligaseat 14° C. (for “blunt end” ligation). Intermolecular “sticky end”ligations are usually performed at 30-100 μg/ml total DNA concentrations(5-100 nM total end concentration). AAV vectors which contain ITRs.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York.Particularly suitable transfection methods include calcium phosphateco-precipitation, direct micro-injection into cultured cells,electroporation, liposome mediated gene transfer, lipid-mediatedtransduction, and nucleic acid delivery using high-velocitymicroprojectiles.

In one embodiment, suitable host cells for producing rAAV virionsinclude microorganisms, yeast cells, insect cells, and mammalian cells,that can be, or have been, used as recipients of a heterologous DNAmolecule. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. Cellsfrom the stable human cell line, 293 (readily available through, e.g.,the American Type Culture Collection under Accession Number ATCCCRL1573) can be used in the practice of the present disclosure.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments, andexpresses the adenoviral Ela and E1b genes. The 293 cell line is readilytransfected, and provides a particularly convenient platform in which toproduce rAAV virions.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. Suitable homologues of the AAV rep coding region include thehuman herpesvirus 6 (HHV-6) rep gene which is also known to mediateAAV-2 DNA replication.

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome.

In one embodiment, AAV helper functions are introduced into the hostcell by transfecting the host cell with an AAV helper construct eitherprior to, or concurrently with, the transfection of the AAV expressionvector. AAV helper constructs are thus used to provide at leasttransient expression of AAV rep and/or cap genes to complement missingAAV functions that are necessary for productive AAV infection. AAVhelper constructs lack AAV ITRs and can neither replicate nor packagethemselves. These constructs can be in the form of a plasmid, phage,transposon, cosmid, virus, or virion. A number of AAV helper constructshave been described, such as the commonly used plasmids pAAV/Ad andpIM29+45 which encode both Rep and Cap expression products. A number ofother vectors have been described which encode Rep and/or Cap expressionproducts.

Methods of delivery of viral vectors include injecting the AAV2 into theCSF. Generally, rAAV virions may be introduced into cells of the CNSusing either in vivo or in vitro transduction techniques. If transducedin vitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

In one embodiment, pharmaceutical compositions will comprise sufficientgenetic material to produce a therapeutically effective amount of thenucleic acid of interest, i.e., an amount sufficient to reduce orameliorate symptoms of the disease state in question or an amountsufficient to confer the desired benefit. The pharmaceuticalcompositions will also contain a pharmaceutically acceptable excipient.Such excipients include any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,sorbitol, Tween80, and liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. A thorough discussion of pharmaceutically acceptableexcipients is available in Remington's Pharmaceutical Sciences (MackPub. Co., N.J. 1991).

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present disclosure becombined with other suitable compositions and therapies.

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

In certain embodiments, the rAAV is administered at a dose of about 1-5ml of 1×10⁵ -1×10¹⁶ vg/ml. In certain embodiments, the rAAV isadministered at a dose of about 1-3 ml of 1×10⁷-1×10¹⁴ vg/ml. In certainembodiments, the rAAV is administered at a dose of about 1-2 ml of1×10⁸-1×10¹³ vg/ml.

Formulations containing the rAAV particles will contain an effectiveamount of the rAAV particles in a vehicle, the effective amount beingreadily determined by one skilled in the art. The rAAV particles maytypically range from about 1% to about 95% (w/w) of the composition, oreven higher or lower if appropriate. The quantity to be administereddepends upon factors such as the age, weight and physical condition ofthe animal or the human subject considered for treatment. Effectivedosages can be established by one of ordinary skill in the art throughroutine trials establishing dose response curves. The subject is treatedby administration of the rAAV particles in one or more doses. Multipledoses may be administered as is required to maintain adequate enzymeactivity.

Vehicles including water, aqueous saline, artificial CSF, or other knownsubstances can be employed with the subject invention. To prepare aformulation, the purified composition can be isolated, lyophilized andstabilized. The composition may then be adjusted to an appropriateconcentration, optionally combined with an anti-inflammatory agent, andpackaged for use.

TPP1 Protein

In certain embodiments, the nucleic acid being administered encodesTPP1, a TPP1 that has substantial identity to wildtype TPP1, and/or avariant, mutant or fragment of TPP 1. The human TPP1 amino acid sequenceis provided in FIG. 14, and the nucleic acid sequence is provided inFIGS. 15A and 15B. FIG. 16 provides the Macaca mulatta TPP1 amino acidsequence, and FIG. 17 provides the Macaca fascicularis TPP1 amino acidsequence. In certain embodiments, the TPP1 protein can have about 70%homology, about 75% homology, 80% homology, 85% homology, 90% homology,95% homology, 98% homology, 99% homology, or even 100% homology to theprotein set forth in FIG. 14, 16 or 17. The TPP1 protein can have about70% identity, about 75% identity, 80% identity, 85% identity, 90%identity, 95% identity, 98% identity, 99% identity, or even 100%identity to the protein set forth in FIG. 14, 16 or 17.

A mutant protein refers to the protein encoded by a gene having amutation, e.g., a missense or nonsense mutation in TPP1. The term“nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, composed ofmonomers (nucleotides) containing a sugar, phosphate and a base that iseither a purine or pyrimidine. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues.

A “nucleic acid fragment” is a portion of a given nucleic acid molecule.Deoxyribonucleic acid (DNA) in the majority of organisms is the geneticmaterial while ribonucleic acid (RNA) is involved in the transfer ofinformation contained within DNA into proteins. Fragments and variantsof the disclosed nucleotide sequences and proteins or partial-lengthproteins encoded thereby are also encompassed by the present invention.By “fragment” or “portion” is meant a full length or less than fulllength of the nucleotide sequence encoding, or the amino acid sequenceof, a polypeptide or protein. In certain embodiments, the fragment orportion is biologically functional (i.e., retains 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or 100% of enzymatic activity of the wildtype TPP1).

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis, which encode the native protein, as well as those thatencode a polypeptide having amino acid substitutions. Generally,nucleotide sequence variants of the invention will have at least 40%,50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequenceidentity to the native (endogenous) nucleotide sequence. In certainembodiments, the variant is biologically functional (i.e., retains 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or 100% of enzymatic activity of the wildtypeTPP1).

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences. Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given polypeptide. For instance, the codons CGT, CGC, CGA,CGG, AGA and AGG all encode the amino acid arginine. Thus, at everyposition where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described without alteringthe encoded protein. Such nucleic acid variations are “silentvariations,” which are one species of “conservatively modifiedvariations.” Every nucleic acid sequence described herein that encodes apolypeptide also describes every possible silent variation, except whereotherwise noted. One of skill in the art will recognize that each codonin a nucleic acid (except

ATG, which is ordinarily the only codon for methionine) can be modifiedto yield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid that encodes apolypeptide is implicit in each described sequence.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%,or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, comparedto a reference sequence using one of the alignment programs describedusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning, and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of at least 70%, atleast 80%, 90%, or even at least 95%.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even, 95%,96%, 97%, 98% or 99%, sequence identity to the reference sequence over aspecified comparison window. An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution.

Apolipoprotein E (ApoE)

There are several different human apolipoprotein E (ApoE) isoforms, thepresence of some of these isoforms in the brain increase the risk forAlzheimer's disease (AD), whereas the presence of other isoformsdecreases the risk for AD. The presence of the ApoE c4 isoform is astrong genetic risk factor for late-onset, sporadic AD. (Casellano etal., Sci Transl Med, 3(89):89ra57 (29 Jun. 2011).) The ApoE ϵ4 allelestrongly increases AD risk and decreases age of onset. On the otherhand, the presence of the ApoE ϵ2 allele appears to decrease AD risk. Itis suggested that human ApoE isoforms differentially affect theclearance or synthesis of amyloid-β (Aβ) in vivo.

In certain embodiments, the nucleic acid being administered encodesApoE, a ApoE that has substantial identity to wildtype ApoE, or avariant, mutant and/or or fragment of ApoE. In certain embodiments, thenucleic acid encodes ApoE ϵ2, an ApoE ϵ2 that has substantial identityto wildtype ApoE ϵ2, and/or a variant, mutant or fragment of ApoE ϵ2.

Immunesuppression Agents

In certain embodiments, an immunesuppression agent is also administeredto the mammal. In certain embodiments, the immuesuprression agent is ananti-inflammatory agent. In certain embodiments, the anti-inflammatoryagent is mycophenolate. In certain embodiments, the anti-inflammatoryagent is administered prior to the administration of the rAAV particles.In certain embodiments, the anti-inflammatory agent is administeredconcurrently to the administration of the rAAV particles. In certainembodiments, the anti-inflammatory agent is administered subsequent tothe administration of the rAAV particles.

In certain embodiments, the anti-inflammatory agent is administeredparenterally, such as by intramuscular or subcutaneous injection in anappropriate vehicle. Other modes of administration, however, such asoral, intranasal or intradermal delivery, are also acceptable. Incertain embodiments, a composition comprising the rAAV particle and theanti-inflammatory agent is prepared and the anti-inflammatory agent andrAAV particle are administered simultaneously to the mammal's cisternamagna and/or to the mammal's brain ventricle, subarachnoid space and/orintrathecal space.

Methods for Introducing Genetic Material into Cells

The exogenous genetic material (e.g., a cDNA encoding one or moretherapeutic proteins) is introduced into the cell in vivo by genetictransfer methods, such as transfection or transduction, to provide agenetically modified cell. Various expression vectors (i.e., vehiclesfor facilitating delivery of exogenous genetic material into a targetcell) are known to one of ordinary skill in the art.

As used herein, “transfection of cells” refers to the acquisition by acell of new genetic material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid into a cell usingphysical or chemical methods. Several transfection techniques are knownto those of ordinary skill in the art including: calcium phosphate DNAco-precipitation; DEAE-dextran; electroporation; cationicliposome-mediated transfection; and tungsten particle-faciliatedmicroparticle bombardment. Strontium phosphate DNA co-precipitation isanother possible transfection method.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. A RNAvirus (i.e., a retrovirus) for transferring a nucleic acid into a cellis referred to herein as a transducing chimeric retrovirus. Exogenousgenetic material contained within the retrovirus is incorporated intothe genome of the transduced cell. A cell that has been transduced witha chimeric DNA virus (e.g., an adenovirus carrying a cDNA encoding atherapeutic agent), will not have the exogenous genetic materialincorporated into its genome but will be capable of expressing theexogenous genetic material that is retained extrachromosomally withinthe cell.

Typically, the exogenous genetic material includes the heterologous gene(usually in the form of a cDNA comprising the exons coding for thetherapeutic protein) together with a promoter to control transcriptionof the new gene. The promoter characteristically has a specificnucleotide sequence necessary to initiate transcription. Optionally, theexogenous genetic material further includes additional sequences (i.e.,enhancers) required to obtain the desired gene transcription activity.For the purpose of this discussion an “enhancer” is simply anynon-translated DNA sequence which works contiguous with the codingsequence (in cis) to change the basal transcription level dictated bythe promoter. The exogenous genetic material may introduced into thecell genome immediately downstream from the promoter so that thepromoter and coding sequence are operatively linked so as to permittranscription of the coding sequence. A retroviral expression vector mayinclude an exogenous promoter element to control transcription of theinserted exogenous gene. Such exogenous promoters include bothconstitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a gene under the control of aconstitutive promoter is expressed under all conditions of cell growth.Exemplary constitutive promoters include the promoters for the followinggenes which encode certain constitutive or “housekeeping” functions:hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase(DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvatekinase, phosphoglycerol mutase, the actin promoter, and otherconstitutive promoters known to those of skill in the art. In addition,many viral promoters function constitutively in eucaryotic cells. Theseinclude: the early and late promoters of SV40; the long terminal repeats(LTRs) of Moloney Leukemia Virus and other retroviruses; and thethymidine kinase promoter of Herpes Simplex Virus, among many others.Accordingly, any of the above-referenced constitutive promoters can beused to control transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressedonly or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). Induciblepromoters include responsive elements (REs) which stimulatetranscription when their inducing factors are bound. For example, thereare REs for serum factors, steroid hormones, retinoic acid and cyclicAMP. Promoters containing a particular RE can be chosen in order toobtain an inducible response and in some cases, the RE itself may beattached to a different promoter, thereby conferring inducibility to therecombinant gene. Thus, by selecting the appropriate promoter(constitutive versus inducible; strong versus weak), it is possible tocontrol both the existence and level of expression of a therapeuticagent in the genetically modified cell. If the gene encoding thetherapeutic agent is under the control of an inducible promoter,delivery of the therapeutic agent in situ is triggered by exposing thegenetically modified cell in situ to conditions for permittingtranscription of the therapeutic agent, e.g., by intraperitonealinjection of specific inducers of the inducible promoters which controltranscription of the agent. For example, in situ expression bygenetically modified cells of a therapeutic agent encoded by a geneunder the control of the metallothionein promoter, is enhanced bycontacting the genetically modified cells with a solution containing theappropriate (i.e., inducing) metal ions in situ.

Accordingly, the amount of therapeutic agent that is delivered in situis regulated by controlling such factors as: (1) the nature of thepromoter used to direct transcription of the inserted gene, (i.e.,whether the promoter is constitutive or inducible, strong or weak); (2)the number of copies of the exogenous gene that are inserted into thecell; (3) the number of transduced/transfected cells that areadministered (e.g., implanted) to the patient; (4) the size of theimplant (e.g., graft or encapsulated expression system); (5) the numberof implants; (6) the length of time the transduced/transfected cells orimplants are left in place; and (7) the production rate of thetherapeutic agent by the genetically modified cell. Selection andoptimization of these factors for delivery of a therapeuticallyeffective dose of a particular therapeutic agent is deemed to be withinthe scope of one of ordinary skill in the art without undueexperimentation, taking into account the above-disclosed factors and theclinical profile of the patient.

In addition to at least one promoter and at least one heterologousnucleic acid encoding the therapeutic agent, the expression vector mayinclude a selection gene, for example, a neomycin resistance gene, forfacilitating selection of cells that have been transfected or transducedwith the expression vector. Alternatively, the cells are transfectedwith two or more expression vectors, at least one vector containing thegene(s) encoding the therapeutic agent(s), the other vector containing aselection gene. The selection of a suitable promoter, enhancer,selection gene and/or signal sequence (described below) is deemed to bewithin the scope of one of ordinary skill in the art without undueexperimentation.

The therapeutic agent can be targeted for delivery to an extracellular,intracellular or membrane location. If it is desirable for the geneproduct to be secreted from the cells, the expression vector is designedto include an appropriate secretion “signal” sequence for secreting thetherapeutic gene product from the cell to the extracellular milieu. Ifit is desirable for the gene product to be retained within the cell,this secretion signal sequence is omitted. In a similar manner, theexpression vector can be constructed to include “retention” signalsequences for anchoring the therapeutic agent within the cell plasmamembrane. For example, all membrane proteins have hydrophobictransmembrane regions, which stop translocation of the protein in themembrane and do not allow the protein to be secreted. The constructionof an expression vector including signal sequences for targeting a geneproduct to a particular location is deemed to be within the scope of oneof ordinary skill in the art without the need for undue experimentation.

EXAMPLE 1 Methods of Gene Transfer in Large Mammals

Lysosomal storage disorders (LSDs) constitute a large class of inheritedmetabolic disorders. Most LSDs are caused by lysosomal enzymedeficiencies which lead to organ damage and often central nervous system(CNS) degeneration. Late infantile neuronal ceroid lipofuscinosis(LINCL) is an autosomal recessive neurodegenerative disease caused bymutations in a ceroid-lipofuscinosis (CLN), neuronal 2 gene CLN2, whichencodes the lysosomal protease tripeptidyl peptidase 1 (TPP1). LINCL ischaracterized clinically by normal birth and early development, onset ofseizures by 18-24 months, progressive motor and cognitive decline, andpremature death. The disease is due to a deficiency in TPP1, which is asoluble, M-6-P decorated lysosomal enzyme.

Enzyme-replacement therapy (ERT) is currently available for lysosomalstorage diseases affecting peripheral tissues, but has not been used inpatients with central nervous system (CNS) involvement. A recent studyinvestigated whether enzyme delivery through the cerebrospinal fluid wasa potential alternative route to the CNS for LINCL (Chang et al.,Molecular Therapy 16:649-656, 2008). Treated mice showed attenuatedneuropathology, and decreased resting tremor relative to vehicle-treatedmice.

In the present work, it was investigated whether global delivery of avector could be effectively performed in order to achieve steady-statelevels of enzyme in the cerebrospinal fluid (CSF) by means of injectionin the brain. Studies were performed in a dog model of LINCL.

The LINCL dogs are normal at birth, but develop neurological signsaround 7 months, testable cognitive deficits at ˜5-6 months, seizures at10-11 months, and progressive visual loss. The CLN2 gene mutation in theLINCL dog renders the TPP1 protein non-functional, and TPP1 protein isundetectable. With disease progression, brain tissues shrink, leading toenlarged ventricular spaces in the brain. Neurological symptoms includedecline in balance and motor functions, loss of vision, tremors.

Affected LINCL pups were given gene therapy at three months of age. Forgene therapy, AAV2-CLN2 generated (see WO 2012/135857), and was injectedat a single site (lateral ventricle) or at two sites (lateral ventricleplus cisterna magna) in the brain. Needles were placed into theventricle, or into the ventricle and cistern magna, and vector infusedslowly over several minutes. While much of the TPP1 made within a cellstayed in that cell, a portion was secreted and taken up by neighboringcells. This property of secretion and uptake is called“cross-correction” (FIG. 2). Cross-correction is valuable in the contextof gene therapy in that if the CLN2 gene is transferred to strategicallysituated cells in the LINCL brain, then this can allow forcross-correction of many surrounding cells.

In the present study, the problem of globally delivering the therapeuticvector took advantage of the CSF flow in the brain by targeting cellsthat line the ventricles and cells that make up the meninges. AAV2-CLN2was injected at a single site (lateral ventricle) or at two sites(lateral ventricle plus cisterna magna) in the brain. TPP1 expressionwas observed in Cln2^(-/-) dogs after AAV delivery (FIG. 3). Asignificant positive impact was observed on ventricular volume. Theeffect of AAV.TPP1 on autofluorescence was also evaluated (FIG. 4A).FIG. 4B shows T1-MM images of untreated and treated dogs. FIG. 4C showsthe effects of AAV.TPP1 in LINCL dogs. FIG. 4D shows huTPP1 distributionafter AAV2/2-huCLN2 administration.

In untreated affected dogs, ventricular spaces enlarge to ten times thesize of normal dogs, whereas AAV2-CLN2 gene therapy significantlyreduced this effect. Further, a broad distribution of enzyme wasobserved, as was a clinical benefit (lifespan and clinical examination).Without treatment, affected dogs show signs of disease in all 22 testsby 30 weeks of age. They reach end-stage disease and must be euthanizedbetween 45 and 48 weeks of age. In dogs that received AAV2-CLN2 genetherapy, the onset of every one of these signs was delayed or prevented.

An increase in TPP1 activity was observed in CSF after combined cisternaand ventricular delivery.

Thus, in the LINCL dog, AAV2-CLN2 gene transfer resulted in TPP1 proteinreplenishment to many areas of the brain, and the results indicated thatAAV2-CLN2 gene transfer provided significant therapeutic effects,reduced or delayed symptoms and improved the quality of life for theLINCL dogs.

The huTPP1 activity in CSF declined shortly after injection (FIG. 5). Abroad distribution of enzyme was observed, but the levels were low atthe time of sacrifice 6-8 months post-gene therapy. It was postulatedthat the decline in activity was a result of an immune response to thehuman enzyme in the dogs. In order to inhibit the decline in activity,an anti-inflammatory agent (mycophenolate) was introduced. The resultsindicated that the anti-inflammatory agent did not inhibit the enzymaticactivity of the huTPP1, and was effective in extending the length oftime that the enzyme activity was present (FIG. 6), and sustained enzymeactivity levels were observed.

High caTPP1 activity in CSF was observed along the time after AAV2caCLN2intraventricular injection and early mycophenolate treatment (FIG. 7).

An increase in TPP1 enzyme activity was observed in many tissues twomonths post-administration (FIGS. 8, 9A and 9B).

FIG. 10 shows the onset of clinical signs in LINCL dogs. caTPP1 activitywas observed in meninges and peripheral tissue, such as the liver (FIG.11).

Thus, the inventors have shown the transformation of pendymal cells byAAV2/2, that canine TPP1 enzyme was produced and flowed with CSF, andthat mycopheolate treatment pro rot caCLN2 injection could preventimmunoresponse in dogs.

EXAMPLE 2 Studies in Non-Human Primates

Using techniques similar to those described above, the inventorsobserved that AAVeGFP transduced ependyma in nonhuman primate brain. Invivo assessment of

AAV2/2.TPP1 delivery in rhesus brain was performed by injecting AAV.TPP1into the ventricle or cisterna magna, harvesting the tissue 4-12 weekslater, and evaluating the TPP1 activity in CSF or tissue lysates (FIG.12). Activity was observed in the vestibular area (brainstem) in thenon-human primates (FIG. 13). Thus, the ventricular lining cellsprovided a source of recombinant enzyme for broad CNS distribution.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A method of delivering a therapeutic agent to the central nervoussystem of a mammal, comprising administering to the mammal's cisternamagna an rAAV particle comprising an AAV capsid protein and a vectorcomprising a nucleic acid encoding a therapeutic agent inserted betweena pair of AAV inverted terminal repeats in a manner effective to infectcells that contact the cerebrospinal fluid (CSF) of the mammal such thatthe cells express the therapeutic agent in the mammal.
 2. A method oftreating a disease in a mammal comprising administering to the mammal'scisterna magna an rAAV particle comprising an AAV capsid protein and avector comprising a nucleic acid encoding a therapeutic agent insertedbetween a pair of AAV inverted terminal repeats in a manner effective toinfect cells that contact the cerebrospinal fluid (CSF) of the mammal,wherein the cell expresses the therapeutic agent so as to treat thedisease.
 3. A method of delivering a therapeutic agent to the centralnervous system of a mammal, comprising administering to the mammal'sbrain ventricle, subarachnoid space and/or intrathecal space an rAAVparticle comprising an AAV capsid protein and a vector comprising anucleic acid encoding a therapeutic agent inserted between a pair of AAVinverted terminal repeats in a manner effective to infect cells thatcontact the cerebrospinal fluid (CSF) of the mammal such that the cellsexpress the therapeutic agent in the mammal.
 4. A method of treating adisease in a mammal comprising administering to the mammal's brainventricle, subarachnoid space and/or intrathecal space an rAAV particlecomprising an AAV capsid protein and a vector comprising a nucleic acidencoding a therapeutic agent inserted between a pair of AAV invertedterminal repeats in a manner effective to infect cells that contact thecerebrospinal fluid (CSF) of the mammal, wherein the cell expresses thetherapeutic agent so as to treat the disease.
 5. The method of claim 1,wherein the cell expresses the therapeutic agent and secretes thetherapeutic agent into the CSF.
 6. The method of claim 1, wherein thecell is an ependymal, pial, endothelial, brain ventricle, and/ormeningeal cell.
 7. The method of claim 1, further comprisingadditionally administering the rAAV to the mammal's brain ventricle,subarachnoid space and/or intrathecal space.
 8. The method of claim 1,wherein the mammal is a non-rodent mammal. 9-10. (canceled)
 11. Themethod of claim 8, wherein the non-rodent mammal is a primate.
 12. Themethod of claim 11, wherein the primate is human.
 13. The method ofclaim 1, wherein the therapeutic agent is a therapeutic nucleic acid.14. The method of claim 1, wherein the therapeutic agent is a protein.15. The method of claim 14, wherein the nucleic acid encodes a lysosomalhydrolase.
 16. The method of claim 15, wherein the protein is TPP1. 17.The method of claim 1, wherein the disease is a lysosomal storagedisease (LSD).
 18. The method of claim 17, wherein the LSD is infantileor late infantile ceroid lipofuscinoses (LINCL), neuronopathic Gaucher,Juvenile Batten, Fabry, MLD, Sanfilippo A, Hunter, Krabbe, Morquio,Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), Sanfilippo B,Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-I H/S),Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten, GM1Gangliosidosis, Mucolipidosis type II/III, or Sandhoff disease.
 19. Themethod of claim 18, wherein the disease is LINCL. 20-25. (canceled) 26.The method of claim 1, wherein the rAAV particle is injected at 1-5locations in the brain.
 27. (canceled)
 28. The method of claim 1,wherein the rAAV particle is an rAAV2, rAAV4, rAAV5 and/or rAAV9particle.
 29. The method of claim 28, wherein the rAAV particle is anrAAV2 particle.
 30. (canceled)
 31. The method of claim 1, wherein thetherapeutic agent is administered in a single dose to the mammal'scisterna magna.
 32. The method of claim 1, further comprisingadministering an immunesuppression agent.
 33. The method of claim 32,wherein the immuesuppression agent is an anti-inflammatory agent. 34.The method of claim 33, wherein the anti-inflammatory agent ismycophenolate.
 35. The method of claim 1, wherein the rAAV isadministered at a dose of about 1-5 ml of 1×10⁵-1×10¹⁶ vg/ml. 36-37.(canceled)
 38. The method of claim 2, further comprising additionallyadministering the rAAV to the mammal's brain ventricle, subarachnoidspace and/or intrathecal space.